Transcript pres_jillian - Harlem Children Society

The Study of Triazine
Antimicrobial Compounds
Jillian Greenaway
New York University
Dr. Neville Kallenbach
Objective
The goal of this project is to synthesize
Triazine compounds using biomimetics
and test how well they inhibit the
growth of Bacillus Subtilis.
Problem
Peptides, when used in our bodies to
kill bacteria, can be digested by protein
digesting enzymes. In a result of this,
we had to think of another strategy to
inhibit bacterial growth. This is when
we thought of biomimetics.
Biomimetics
Biomimetics also known as Bionics, is
the implementation of methods found in
nature to the study of modern
technology. We used biomimetics,
specifically to make a synthetic material
mimic a naturally occurring substance
by giving it positively charged and
hydrophobic properties.
Hypothesis
Using biomimetics, an organic molecule
can act the same way as a protein with
similar properties. The properties for
antimicrobial activity are an overall 2+
charge and 2 bulky hydrophobic groups.
If enough of this compound with these
characteristics is associated with the
membrane, it can break the membrane
and kill the bacteria.
Triazine Libraries
The lab workers synthesized triazine
libraries to test on Bacillus Subtilis by
using Combinatorial Chemistry, which is
the act of mixing products. Libraries are
groups of the same compound
synthesized in various ways. The
following charts depict the two groups
of triazine libraries used.
Group 1 Compounds
R1
R1
N
N
HO
A
B
HN
N
Structure
R2
This plate was
chosen for the
many bulky
hydrophobic
groups. The third
functional group
used in this library
is adamantane.
3
NH2
HO
D
HO
NH2
NH2
HO
OH
NH2
HO
H
5
6
NH2
8
O
NH2
Bz
H2N
H2N
H2N
OH
H2N
H2N
H2N
OMe
O
H
N
H2N
7
OH
HO
H2N
4
NH2
G
I
J
1
2
NH2
NH2
C
E
F
R2 Structure
O
O
NH2
Positive Charge – Group 2
R1 Aryl/Alkyl
NH
O
O
HN
A
N
N
B
R1
N
R2
C
This group was
chosen to have
D
sufficient positive
E
charge and also,
hopefully, to have F
enough
G
hydrophobic bulk
H
to be active.
2
Cl
R2
1
2
3
4
5
6
7
8
9
Amine
N
NH2
N
N
H
NH2
N
O
N
H2N
N
NH2
N
H2N
N
H2N
N
H2N
HN
N
OH
Methods and Materials
In order to determine which compound
kills bacteria efficiently, we ran assays
or tests.
1. First we grew the bacteria in an
incubator shaker for several hours.
2. Secondly, we set up plates containing
twenty-four wells.
Each well contained 800 microliters of
plain media, Bacillus Subtilis, sodium
phosphate buffer, and a different
concentration of the triazine compound.
The amount of phosphate buffer and
compound together equaled 150
microliters. All the wells contained 50
microliters of cell culture, which
included Bacillus Subtilis and Tryptic
Soy Broth. I allowed all the plates to
grow for six hours.
3. After six hours I ran all the samples
through the spectrophotometer. With a
push a button we were presented with
the samples’ light absorbance in a
numerical value. I took this number and
computed it into the following equation.
1- (Average Absorbance from the
compound/ Average absorbance from the
control) X 100
This equation results in the inhibition
percentage. Therefore, if the
percentage is high then the compound
worked efficiently. On the other hand, if
the percentage inhibition is low, the
compound did not stop the bacteria
from growing.
Results from Group 1
% Inhibition at 100mM
1
2
3
4
5
6
7
8
A
B
C
*
73.1
4.1
*
99.4 88.5
D
E
77.3 54.2
*
F
4.3
G
H
I
J
38.3 28.0 40.8 44.3
29.3 84.1 33.7 96.6 44.9 39.2
3.9
19.1 42.0 11.5
*
0.7
39.2
*
44.0
9.3
*
*
33.9 26.3
7.5
*
1.5
10.5 23.9 94.1
3.9
*
5.5
97.2 23.7 17.1 29.9
50.1
8.1
98.1 69.1 22.3 99.6 99.6
5.6
97.2 82.5 97.8 12.6 10.5
72.4 67.7
* = 0% Inhibition
*
27.8 27.4
9.2
6.7
44.7 30.2
*
*
15.6 18.3
7.2
100
15.2 10.4
*
7.6
4.4
Positive Results from Group 1
1
2
3
4
5
6
7
8
A
B
C
*
73.1
4.1
*
99.4 88.5
D
E
77.3 54.2
*
F
4.3
G
H
I
J
38.3 28.0 40.8 44.3
29.3 84.1 33.7 96.6 44.9 39.2
3.9
19.1 42.0 11.5
*
0.7
39.2
*
44.0
9.3
*
*
33.9 26.3
7.5
*
1.5
10.5 23.9 94.1
3.9
*
5.5
97.2 23.7 17.1 29.9
50.1
8.1
98.1 69.1 22.3 99.6 99.6
5.6
: >90% inhibition
97.2 82.5 97.8 12.6 10.5
72.4 67.7
*
27.8 27.4
9.2
6.7
44.7 30.2
*
*
15.6 18.3
7.2
100
15.2 10.4
*
7.6
4.4
: >80% inhibition
Results from Group 2
% Inhibition at 100mM
1
2
3
4
5
6
7
8
9
A
5.6
8.1
0.4
*
5.5
9.7
99.7
9.6
4.6
B
99.6
6.2
14.2
4.4
8.5
*
99.5
*
*
C
84.3
15.2
83.4
21.3
100
32.6
99.9
10.6
72.1
D
23.6
16.4
15.0
12.5
7.3
*
*
8.3
6.2
E
99.0
46.7
97.9
78.5
97.9
98.4
98.1
*
7.6
F
85.2
10.6
99.0
*
97.3
*
98.9
1.4
*
G
*
*
*
*
1.0
1.8
*
*
*
H
15.8
2.0
6.3
*
7.1
*
99.4
*
28.6
Results from Group 2
Positive Results Highlighted
1
2
3
4
5
6
7
8
9
A
5.6
8.1
0.4
*
5.5
9.7
99.7
9.6
4.6
B
99.6
6.2
14.2
4.4
8.5
*
99.5
*
*
C
84.3
15.2
83.4
21.3
100
32.6
99.9
10.6
72.1
D
23.6
16.4
15.0
12.5
7.3
*
*
8.3
6.2
E
99.0
46.7
97.9
78.5
97.9
98.4
98.1
*
7.6
F
85.2
10.6
99.0
*
97.3
*
98.9
1.4
*
G
*
*
*
*
1.0
1.8
*
*
*
H
15.8
2.0
6.3
*
7.1
*
99.4
*
28.6
: >90% inhibition
: >80% inhibition
Frequency of Effectiveness (G1)
R1
A
B
C
D
E
F
G
H
I
J
Structure
HO
NH2
NH2
NH2
HO
NH2
HO
NH2
NH2
HO
OH
NH2
HO
OH
HO
NH2
O
NH2
O
Bz
H
N
O
O
NH2
Freq. R2
1
0
2
1
3
3
4
0
5
1
6
3
7
2
8
3
0
0
Structure
H2N
H2N
H2N
H2N
H2N
OH
H2N
H2N
H2N
OMe
Freq.
0
4
3
0
0
1
2
3
Frequency of Effectiveness (G2)
R1
A
B
C
D
E
F
G
H
Structure
Cl
Freq. R2
1
1
2
2
3
4
4
0
5
5
6
4
7
0
8
1
9
Structure
N
NH2
N
N
H
NH2
N
O
N
H2N
N
NH2
N
H2N
N
H2N
N
H2N
HN
N
OH
Freq.
4
0
3
0
3
1
6
0
0
Analysis
Group one did not have much positive results
because of the lack of positive charge in the
library.
Group 2 had more positive results than Group
one because it had both positively charged
and hydrophobic groups. But it is possible
that we’ve gone too far in the other direction
and that there is now enough positive charge,
but no longer sufficient hydrophobic bulk.
Conclusion
We analyzed the data and designed a
new library.
The following chart depicts the functional
groups for these libraries.
“Desirable” Functional Groups
Hydrophobic Groups:
Plate 1, # 2:
H2N
Plate 1, # 3:
H2N
Plate 1, # 8:
Plate 1, # C:
Plate 1, # F:
Plate 1, # H:
Charged Groups:
N
Plate 2, # 7:
H2N
H2N
OMe
Plate 2, # 1:
N
NH2
NH2
HO
HO
HO
NH2
OH
Plate 2, # 5:
NH2
N
NH2
O
Plate 2, # C:
Plate 2, # E:
Plate 2, # F:
Cl
Plate 2, # 3:
N
NH2
Proposed New Library
R1
In the new library, we
would like to use a
triazine with
adamantane, then
use the selected
positive groups for
the R1 position, and
cross them with both
the hydrophobic and
charged moieties in
the R2 position. This
should help us to
determine the best
balance of charge vs.
hydrophobicity.
Structure
R2
A
N
A
H2N
Structure
N
H2N
B
N
NH2
C
N
B
NH2
N
NH2
NH2
N
D
1
C
N
NH2
NH2
N
D
2
3
Cl
4
5
6
HN
N
R1
7
N
N
8
R2
9
H2N
H2N
OMe
HO
NH2
O
HO
HO
H2N
NH2
OH
NH2